Project Summary: Cell-free protein synthesis (CFPS) is a method of producing proteins outside living cells and has distinct advantages over traditional cell-culture-based protein production. It is often difficult to maintain complex environmental condition for cell viability and the subsequent need to develop a downstream process for purification of product in traditional cell-based systems. CFPS, on the other hand, can be accomplished just by mixing genetic template (DNA, or mRNA) with a reaction solution containing transcription, translation components (e.g. ribosomes) and nutrient molecules in an in-vitro setup. The synthesis time is also less than a day compared to weeks for the cell-based system. Moreover, lack of cell membrane and open nature of CFPS system can be advantageously used for adding different molecules to optimize the protein synthesis. Consequently, CFPS is used for industrial scale therapeutic protein production, enzyme engineering, protein labeling and studying protein-protein interaction. While the CFPS system has been around for a few decades and has high potential to transform the biosynthesis market, it still has some important issues like high reagent cost, low protein yield, protein aggregation and lack of post-translational modification that has throttled its growth. In this project, we will develop a universal CFPS device that will be able to synthesize proteins at a higher yield, low cost of reagents and even less time to synthesize (1.5 hours compared to 4-6 hours). Dasfanh Bioscience will use patent pending technology to solve the problem by introducing a membrane between reaction and feeding solution, and continuously supplying the nutrients to the reaction chamber and removing by-products from the reaction chamber. This results in significant increase in the protein synthesis yield (one to two orders of magnitude), with a reduction in cost by at least one order of magnitude. Specific Aims: To develop a 96-well-based microfluidic device that can be universally used for all forms of cell free protein synthesis with significantly higher yield in less time. Task 1: Development of 96-well-based continuous exchange CFPS device for high throughput applications Milestone 1: Design and develop the flexible membrane devices for maximum exchange of molecules aided by active transport of fluid. Milestone 2: Extensive testing of commercially available CFPS reagents (both prokaryotic and eukaryotic) to validate efficiency and compatibility with the device. Task 2: Demonstration of versatile usability of the system by co-expressing various proteins and studying interactions among them. Task 3: Adaptation of this technology for co-generation of ribosomal proteins for synthetic biology